EVALUATION OF ATTRIBUTES THAT AFFECT LONGISSIMUS MUSCLE TENDERNESS IN BOS TAURUS AND

BOS /ND/CUS CATTLE192

G. whipple3, M. Koohmaraie4, M. E. dike ma^^^,^, J. D. Crouse4, M. C. Hunt3 and R. D. Klemm6

Kansas State University, Manhattan 66506 and U.S. Department of Agriculture, Clay Center, NE 68933

ABSTRACT

Biological tenderness differences between longissimus muscles (LM) from Bos indicus and Bos fuurus breeds were evaluated. Steers and heifers of Hereford x Angus (H x A, n = lo), 3/8 Sahiwal x H, A or H x A (3/8 SAH, n = 6) and 5/8 Sahiwal x H, A or H x A (5/8 SAH, n = 11) crosses were utilized. Muscle temperature and pH were monitored every 3 h for the first 12 h and at 24 h. Samples were obtained within 1 h and at 24 h postmortem from the LM for determination of calcium-dependent protease (CDP) -I and -11 and CDP inhibitor 0 activities. At 1 and 14 d postmortem, LM samples were removed for determining cathepsin B and B + L activity, soluble and total collagen, sarcomere length, muscle-fiber histochemistry, shear force and sensory-panel traits. Data were analyzed using least squares procedures with fixed effects of breed cross, sex and their interaction. No significant breed cross effects were observed for carcass traits or rates of pH and temperature decline. Steaks from H x A had lower (P < .05) shear-force values and higher (P < .05) sensory scores for tenderness at 1 and 14 d postmortem than steaks from 3/8 and 5/8 SAH. Correspondingly, 5/8 SAH had lower (P < .05) myofibril fragmentation indices than H x A at 1, 3, 7 and 14 d postmortem. Breed cross effects were not significant for sarcomere length, fiber types, soluble and total collagen, cathepsin €3 and B + L specific activity, CDP-I and -II activity and INH activity within 1 h postmortem. However, INH total activity/lOo g of muscle was greater (P < .01) at 24 h postmortem for 5/8 SAH (208.8 f 14.8) and 3/8 SAH (195.6 f 19.3) than for H x A (136.3 f 14.9). For H x A, SDS-PAGE revealed that by d 1 desmin had been subjected to proteolysis, and by d 14 desmin could not be detected, but a 30,000-dalton component was clearly evident. However, in 5/8 SAH, desmin remained visible at d 14 without a 30,000-dalton component appearing. This reduced protein hydrolysis may account for less tender meat in S A H , INH apparently influences this process. (Key Words: Cattle, Tenderness, Proteases, Histochemistry, Protein Degradation, Aging.)

J. Anim. Sci. 1990. 68:271&2728

introduction

'Contribution no. 90-33-J from the Kansas Agric. Ekp.

3 ~ ~ t h ~ ~ wish to thank ~a thy ~ihm, by meer, pa

Cattle producers utilize pro- grams to take advantage of hybrid vigor. Bos

sta. Manhaltan.

Tanrmen and Jill Comer of U.S. Meat Anim. Res. Center and indicus breeds are used frequently to maximize

cal climates where these breeds provide addi- ger Weir of KSU for the typing of this manuscript.

tive advantages for heat and disease resistance USDA, Clay Center, NE. (Cole et al.. 1963: Crockett et al.. 1979).

S a y Stroda Of KSU for their hborato~ aSSktanCe and Gin- hderoSis, especially in semitropical and tropi- b p t . of Anim. Sci. and Ind., Kansas State Univ.

L. U.S. Meat m. Res. cater, ms, 5T0 whom reprint requests should be addressed. 6Dept. of Anat. and F'hysiol., Kansas State Univ. Received September 21,1989. Accepted January 29,1990.

However, m& from Bos indicus breeds o f cattle often is less tender than meat from Bos faurus cattle (Koch et al., 1982; McKeith et

2716

BOS TAURUS AND BOS INDICUS BEEF TENDERNESS 2717

al., 1985b; Crouse et al., 1987, 1989). Because tenderness is a major palatability trait that determines consumer acceptability, it is impor- tant to understand what causes meat from these animals to be less tender.

There have been many attempts to explain the variation in beef tenderness. The effects of animal age (Davis et al., 1979), breed (Koch et al., 1982), nutritional regimen (Dikeman et al., 1985), days fed high-concentrate diets (Taturn et al., 1980) and the use of growth promotants (Unruh et al., 1986) on meat tenderness have been explored. In such cases, factors that influence tenderness may include USDA qual- ity grade, USDA yield grade, rates of pH and temperature decline, sarcomere length, total and soluble collagen, muscle histochemical traits, and activity of proteases thought to be involved in postmortem tenderization (cal- ciumdependent protease and cathepsins). These variables all have some relationship to tenderness; however, most studies evaluating tenderness variation have been narrowly fo- cused. Our objectives were to examine all of these biological traits in an attempt to explain why meat from Bos indicus crossbred cattle is less tender than meat from Bos taurus cattle.

Materials and Methods

Experimental Animals, Seven heifers and four steers of 5/8 Sahiwal x Hereford (H), Angus (A) or H x A ( 9 8 SAH), three heifers and three steers of 3/8 Sahiwal x H, A or H x A (3/8 SAH) and five heifers and five steers of H x A crosses were utilized. Calves were weaned at 6 to 8 mo of age and fed an alfalfa haylage and corn silage diet for 4 mo. Cattle then were fed a corn and corn silage finishing diet until 15 to 17 mo of age. Cattle were selected randomly and slaughtered (three to four animals each week for a 7-wk period). This was done so that enough time and instruments would be available to assay for calcium-dependent protease activity on fresh muscle samples. Within 1 h postmortem, longissimus muscle (LM) samples were taken from alternate sides at the fifth to sixth rib region. Alternate sides were utilized for samp- ling to account for any variation that may occur in rate of temperature decline because of different amounts and positions of kidney and pelvic fat. Carcasses were not electrically stimulated but were chilled at -1'C for 24 h. Carcass data (USDA, 1976) were collected; the

semitendinosus (ST; center portion) and LM (sixth rib to the fifth lumbar region) muscles were removed from the same sides that were previously sampled.

Temperature and pH Determinations. Tem- perature of the LM and ST and pH of the LM were determined at < 1, 3, 6, 9, 12 and 24 h postmortem. The procedure of Bendall (1973) was used to determine pH on 2.5 g of tissue. Temperature of the LM was monitored using an electronic digital probe at the 11th rib region. Three temperature measurements were recorded at approximately 5.7, 2.5 and .3 cm depths from the dorsal surface of the LM, and an average temperature was calculated. Tem- peratures of the ST were determined by a thermometer located in the center of the muscle for 24 h.

Calcium-Dependent Protease and Inhibitor Activities. Activities of calcium-dependent pro- tease (CDP)-I and -II and CDP inhibitor (INH) were determined on 100 g of fresh LM at < 1 h and 24 h postmortem according to the procedures of Koohmaraie et al. (1989). Proteins were eluted at the rate of 30 ml/h with an 840-ml linear gradient (0 to 500 mM NaCl in elution buffer; Figure 1). The INH was eluted at approximately 75 mM NaCl (frac- tions 45 to 62), whereas CDP-I was eluted at 125 mM NaCl (fractions 67 to 78); CDP-II was eluted in fractions 105 to 118. Activities were expressed as the amount of CDP caseino- lytic activity in 100 g of muscle. One unit of DEAE-sephacel-pdied-CDP-I and -II activity was defied as the amount of enzyme that catalyzed an increase of 1.0 absorbance unit at 278 nm in 1 h at 25'C. One unit of INH activity was defied as the amount that inhibited 1.0 unit of DEAE-sephacel-purified CDP-II activity.

Cathepsin B and B + L Activities. Lysosomal-enriched fractions were prepared from 5 g of LM taken at 1 and 14 d postmortem according to the method of Bechet et al. (1986). Protein concentration of the supernatant was determined according to Markwell et al. (1978). Activities of cathepsins B and B + L were assayed according to procedures of Kirschke et al. (1983), using amino-methylcoumarin as a fluorescent tag on the substrates, Z-Arg-Arg-NMec and Z-Phe- Arg-NMec (where Z = benzyloxycarbonyl and NMec = 4-methyl-7-coumarylamide), with a 30-min incubation at 37°C. Specific activities were expressed as pmole-mg of pro-

2718

1 I

100 li A

z

FRACTION NUMBER

Figure 1. Elution profile of calcium-dependent protease (CDP)-I, CDP-II and inhibitor from DUE-Sephacel column. The bound proteins were eluted at the rate of 30 ml/h with an 840 ml linear gradient of 0 to 400 mM NaCl m elution buffer.

tein-l.min-1. Myojibril Fragmentation Index Measure-

ments. Samples were excised from the LM at 24 h postmortem and were vacuum-aged for 3, 7 and 14 d. Myofibril fragmentation indices (MFI) were determined on fresh muscle at 1, 3, 7 and 14 d postmortem by the method of Culler et al. (1978).

Determination of Water-Soluble Calcium and Zinc. Water-extractable calcium and zinc were determined on 5 g of muscle according to the method of Nakamura (1973a), using specific atomic absorption.

Muscle-Fiber Histochemistry and Fiber Di- ameter Determinations. The simultaneous histochemical procedure of Solomon and Dunn (1988) was slightly modified. Longissimus samples were removed from the center of the muscle at the eighth rib region at 24 h postmortem. Several i-cm3 samples were frozen on cork in isopentane at -147°C and stored at -7o'C. Cryostat sections, 10 p thick, were cut and allowed to air dry. A 45-min, PNADH (pH 7.4) incubation (37'C) was applied prior to a 5-min acid incubation @H 4.1). An ATPase incubation @H 9.4) was conducted for 30 min at 37'C. Fibers were classified as PR, CLR or a W according to Ashmore and Doerr (1971). Fiber areas were measured by Microcomp PM7 interactive image analysis €or planar morphometry. Fiber

7 s ~ ~ t h e m mm Instnunem, ~tlanta, GA.

diameters were calculated from the area measured for each fiber. The area occupied by each of the three fiber types was reported as a percentage of total area measured.

Sarcomere Length Measurements. Sarco- mere lengths were determined on fixed LM obtained at 24 h postmortem using a neon laser according to Cross et al. (1981).

Total and Soluble Collagen Content. Sam- ples of LM were analyzed on d 1 and 14 postmortem €or soluble and insoluble collagen (Hill, 1966). Hydroxyproline content was determined by the method of Bergman and Loxley (1963). Hydroxyproline contents of the soluble and insoluble portions were multiplied by factors of 7.52 and 7.25, respectively, according to Cross et al. (1973). Values are reported per gram of wet tissue.

Muscle Composition. Fat and dry matter percentages were determined on 3 to 5 g of LM taken at 24 h postmortem according to AOAC (1975) procedures.

Myofibril Isolation and SDS-PAGE. My- ofibrils were isolated according to the method of Olson et al. (1976) at e 1 h, 1 and 14 d postmortem; protein concentration was deter- mined by biuret procedures. Electrophoretic techniques of Laemmli (1970) were followed. Myofibrillar proteins were separated using a discontinuous gradient slab gel with 7.5 to 15% acrylamide. The acrylamide to bisacryla- mide ratio was 37.5:l. The stacking gel consisted of 4% acrylamide. Eighty micro-

BOS TAURUS AND BOS INnICUS BEEF

grams of protein were applied to each well and 8 milliamperes (mA) were used per gel until protein samples had focused; then current was increased to 24 mA/gel.

Warner-Bratzler Shear and Sensory-Panel Evaluations. At 24 h postmortem, six steaks, 2.54 cm thick, were removed from the loin beginning at approximately the first lumbar vertebra, vacuum-packaged, aged for the ap- propriate time period (1 or 14 d) and frozen at -30°C. Steaks were thawed and cooked on Farbemare open-hearth broilers to an endpoint temperature of 70°C. For Instron Wamer- Bratzler shear (WBS) determinations, steaks were tempered for approximately 24 h at refrigerator temperature. Six 1.3-cm cores were removed parallel to the muscle fibers with a hand coring device. For sensory-panel-tender- ness scores, cooked steaks were wrapped in foil and held at 70°C (<30 min) in a preheated oven. In four sessions (two slaughter groups/ session), eight panelists evaluated three (1.3 x 1.3 x 2.5 cm) cubes/steak with an 8-point scoring system according to procedures out- lined by AMSA (1978).

Statistical Analysis. Analysis of variance was performed using the General Linear Models procedure of SAS (1985) that included the fixed effects of breed, sex and their interaction. Means were separated by least squares procedures.

Results and Discussion

The cattle utilized had similar (P > .lo) USDA (1976) quality and yield grades, with averages of high Select and 3.2, respectively. Also, no differences (P > .05) were found for lean color; lean firmness; lean texture; matur- ity scores; dressing percentage and percentage of kidney, pelvic and heart fat (data not presented). Least squares means and standard deviations for growth, carcass and LM chemi- cal traits are given in Table 1. Breed cross effects were observed for average daily gain, with 5/8 SAH gaining the slowest (P < .01). The H x A crosses were heavier (P < .05) at slaughter than 5/8 SAH (502 vs 448 kg, respectively). There were no breed cross marbling differences, which contradicts some reports. Cole et al. (1963) reported differences in marbling and carcass grades between Bos taurus and Bos indicus cattle, with marbling scores of high Modest and avg Slight, respec- tively. Crouse et al. (1989) found that as

2719

2720 WHLPPLE ET AL.

TABLE 2. BREED LEAST SQUARES MEANS ( S E ) FOR WARNER-BRA'IZLER SHEAR VALUES, COOKING LOSSES, AND SENSORY-PANEL SCORES OFLONGISSIMUS STEAKS

Traits Breed cross

H x A 3B sA?I 5/8 S A H ~

Day 1 Shear force, kg 7.0 f Sb 9.3 f .7c 9.6 f .5' cooking loss, 96 27.7 f l.Ob 30.0 f 1.3bc 31.6 f 1.v Juicinessa 5.3 f .1 5.2 f .2 5.1 f .1 mvor intensity" 4.6 f .1 4.6 f .1 4.6 f .1 Ease of fragmentationa 4.7 f .ze 3.7 f .2f 3.6 f .2f

Overall tendernessa 4.6 f .2' 3.6 f .gf 3.6 f .2f Connective tissue amounta 4.4 f .2= 3.4 f .2f 3.4 f .2f

Shear force, kg 4.7 f .3b 6.4 f .4' 7.7 f .3d

Flavor intensity" 4.9 f .1 4.7 f .1 4.7 f .1 Ease of fragmentationa 6.0 f .Zb 5.0 f .2' 4.4 f .2d

Overall tendernessa 5.9 f .2b 5.0 f .2' 4.4 f .2d

Day 14

cooking loss, 96 26.1 f 1.0 28.0 f 1.3 29.6 f 1.0 Juicinessa 5.4 f .1 5.3 f .1 4.8 f .1

Connective tissue amounta 5.9 f .2' 4.7 f .2f 4.2 f .2f

'A score of 6 =moderately juicy, moderately intense, moderately easy, traces and moderately tender, . . .4 = slightly dry, slightly bland, slightly difficutt, moderate and slightly tough; . . . 1 = extremely dry, extremely bland, exeemely difficult, abundant and extremely tough.

b7c*dMeans within a row without a common superscript letter differ (P < 03). evfMeans within a row without a common superscript letter differ (P < .Ol).

percentage of Brahman or Sahiwal inheritance increased, carcass weights and marbling decreased. In addition, Bos indicus crosses have been reported to have less tender meat than Bos taurus crosses, even when marbling scores are held constant (Koch et al.. 1988).

A sex effect was observed for adjusted fat thickness; heifers had more (P < .05) fat cover (1.55 vs 1.16 cm) than steers. Significant sex x breed group interactions occurred for slaughter weight, hot-carcass weight, adjusted-fat thick- ness, ribeye area and incidence of heat ring (Table 1). The H x A steers were heavier at slaughter (P e .05; 529 kg) than 318 S A H steers (438 kg) and 5/8 SAH heifers (417 kg); other sex x breed cross groups were intermedi- ate in weight. For hotcarcass weights, 3/8 SAH steer (267 kg) and 5/8 SAH heifer (264 kg) carcasses were lighter (P < .05) than carcasses of H x A steers and 318 SAH heifers. The 3/8 SAH heifers had more (P < .OS) fat cover than the other Bos indicus crosses, with H x A possessing an intermediate amount of finish. The 5/8 S A H heifers and 318 S A H steers had similar ribeye mas, but 5/8 SAH heifer ribeyes were smaller (P < .05) than those from the other breed crosses. The incidence of heat ring occurred more fre- quently in 3/8 SAH steers (lower numeric

score; P < .OS) than in H x A steers and 518 SAH heifers, likely because of less fat cover on 318 SAH steer carcasses.

No breed cross differences (P > .lo) occurred for temperature and pH or tempera- ture with less than 2/10 difference in pH among the breed crosses at any of the time periods (data not presented). Overall rates of temperature decline in LM and ST (Figure 2) show that temperature in ST was initially (< 1 h) lower, however, ST temperature was higher after 6 h postmortem. Twenty-four-hour tem- perahlres of the ST and LM were 4.8 and 1.3'C, respectively. In the LM, 3-h pH was 6.4 and ultimate pH was 5.5 (Figure 3); these are in the normal range (Bouton, 1973).

Sensory panel evaluation scores and WBS values for LM steaks are given in Table 2. The H x A crosses had lower (P e .05) WEiS values and higher (P < .01) sensory panel scores for ease of fragmentation, connective- tissue amount and overall tenderness at 1 and 14 d postmortem. These breed cross differ- ences agree with those reported by Koch et al. (1982). By d 14, 3/8 SAH were more tender than 5/8 SAH, which indicates a greater response to aging. Steaks from H x A and 318 SAH had approximately a 30% decrease in WBS values between 1 and 14 d of aging.

BOS TAURUS AND BOS JNDICUS BEEF TENDERNESS 2721

40 R

6.9 x

0 3 6 9 1 2 1 5 1 8 21 2 3

Time postmortem (h)

Figure2. Overall temperature decline of longissimus and semitendinosus muscles.

0 3 6 9 1 2 1 5 1 8 2 1 2 4

Time postmortem (h)

Figure 3. Longissimus muscle pH decline.

However, WBS values for 5/8 S A H steaks decreased only 20% and were still unaccepta- ble (> 5.0 kg) after 14 d of aging. In addition, 5/8 S A H had a higher (P < .05) cooking loss than H x A at d 1 but no differences were seen at d 14. Our WBS results agree closely with those of Crouse et al. (1989) in which WBS values at 7 d postmortem were 4.4, 5.6, 6.6, and 8.4 kg for H x A, 1/4 SAH, 1/2 S A H and 3/4 SAH crosses, respectively.

Table 3 reveals that breed crosses were similar (P > .05) for semitendinosus steak WBS values and cooking loss at either 1 or 14 d postmortem. Therefore, it appears that tenderness differences between Bos indicus and Bos taurus are greater for certain muscles than for others. However, there was a trend (P = .08) for 5/8 SAH steaks to be less tender at d 14. Dransfield et al. (1981) found that the ST initially was slightly less tender than the LM, but after aging the LM was only half as tender

as the ST. In that same study, a 5°C rise in temperature produced a significant increase in the rate of tenderization. These results indicate that more research needs to be conducted on enzyme activity and its relationship to tender- ness of the ST and other muscles.

Longissimus tenderness is highly correlated with MFI (Olson et al., 1976; Koohmaraie et al., 1987). Figure 4 contains h4FI for LM of breed cross at 1, 3, 7 and 14 d postmortem. The H x A had higher (P < .05) h4FI values than 5/8 S A H at all times. Furthermore, 3/8 SAH had higher (P < -05) MFI values than 5/8 SAH at d 7 and d 14. At d 14, 5/8 S A H had only a slightly larger MFI value than H x A had at d 1 postmortem. This is reflected also in LM WBS values, which were similar at d 14 for 5/8 S A H and d 1 for H x A. Because many changes occur with postmortem aging within the first 24 h (Marsh et al., 1981; Koohmaraie et al., 1987), we could speculate that the LM of H x A already may have undergone

TABLE 3. BREED LEAST SQUARES MEANS (SE) FOR WARNER-BRA'IZLER SHEAR VALUES AND COOKING LOSSES OF SEMITJ5NDINOSUS STEAKSa

Traits

Breed cross H x A 3 B S A H 5/8 S A H

Day 1, shear force, kg 5.8 f .3 6.5 f .4 6.1 f .3 Day 14. shear force, kg 4.4 f .3 4.9 f .3 5.3 f .3 Day 1, cooking loss, % 37.8 f 1.0 38.1 f 1.2 39.9 f .9 Day 14, cooking loss, % 34.8 f 1.4 35.2 f 1.8 37.5 f 1.4

30 differences (P > .05).

2722 WHLPPLE ET AL.

TABLE 4. BREED LEAST SQUARES MEANS (ME) FOR LONGISSIMUS SARCOMERE LENGTH, CALCIUM AND ZINC CONTENT. COLLAGEN AND CATHEPSIN B AND B + L SPECIFIC ACTIVITY"

Breed cross

Traits H x A 3/8 SAH 5/8 S A H Day 1

Sarcomere length, pm 1.83 f .06 1.76 f .08 1.75 f .06

zinc Contenl pglg 9.5 f .5 8.5 f .6 8.9 f .5 Soluble collagen, '3% 13.6 f .6 14.1 f .8 14.2 f .6

2.6 f .1 2.8 f . I Total collagen, mg/g 2.9 f . I cathepsin B, pm01mg-l.min-l 32.4 f 2.9 27.9 f 3.8 34.3 f 2.9 cathepsin B + L, pm01.nq-l m i d 41.7 f 2.7 38.3 f 3.5 44.4 f 2.7

14.1 f 1.4 16.5 f 1.1 Soluble collagen, % 16.7 f 1.2 3.0 f .1 Total collagen, mglg 3.1 f . I 2.7 f .2

cathepsin B. pmol.m&nin-l 30.5 f 1.8 29.0 f 2.5 34.7 f 1.7 cathepsin B + L, pmol.mg-lmin-* 41.5 f 3.3 41.0 f 4.6 46.6 f 3.1

Calcium content, pg/g 10.8 f .I 8.6 f .7 9.5 f .5

Day 14

%No differences (P z .05).

tenderization prior to the 24-h tenderness measurements.

Sarcomere length is related to tenderness, especially in cases of severe shortening (Marsh and Leet, 1966; Harris, 1976). Because no significant sarcomere length differences were observed in our study (Table 4), this factor did not contribute to breed cross differences in tenderness. Sarcomere lengths ranged from 1.75 to 1.83 pm, which are similar to LM sarcomere measurements made by Hunt and Hedrick (1977) and McKeith et al. (1985a).

Likewise, no differences (P > .05) were found among breed crosses for total and percentage of soluble collagen at 1 and 14 d postmortem (Table 4). Therefore, we con- cluded that neither solubility nor quantity of collagen were contributing factors in the tenderness differences observed among breed crosses in our study. Other researchers have found that collagen characteristics were not significantly related to tenderness, especially within the same muscle from animals similar in age (Dransfield, 1977; Seideman et al., 1987). Thus, myofibrillar components may be the major contributor to tenderness variation of the animals in this study, Researchers that have reported a relationship between collagen solu- bility and tenderness (Hall and Hunt, 1982) have looked at treatment effects beyond those in our study.

Differences in the percentages of fiber types and their sizes have been suggested as affecting meat tenderness (Calkins et al., 1981; Ockerman et al., 1984; Seideman and Crouse,

1986). However, we found no differences (P > .05) among breed crosses for fiber type percentage or percentage fiber area (Figure 5). Moody et al. (1970) also found that fiber diameter was not related to tenderness. Melton et al. (1975) reported that red-fiber area was related to animal weight and fat deposition, but not to palatability traits. Therefore, from their results and ours, histological traits did not help explain tenderness differences in LM steaks.

In addition to fiber size and number, the mineral content of different fiber types might be related to tenderness. Of particular interest

.- C '0°1 0 318Sah H x A

1 3 7 1 4

Time postmortem (d)

Figure 4. Longissimus myofibril fragmentation indices at 1,3,7 and 14 dpostmortemby breed cross. Bars without a common superscript letter cliff- (P < .05) within days post- mortem.

BOS TAURUS AND BOS INDICUS BEEF TENDERNESS 2723

50

40 4, 0, m c 30 0,

P)

I

2 n 20

10

0 p4ed a -red a -white

Muscle fiber type

Figure 5. Percentage of f i b m a and fiber type percent- age of longissimas muscle by breed cross.

is ca2+ concentration because of its require ment for CDP activation. The sarcoplasmic reticulum is less developed in red fibers than in white fibers (Gauthier, 1970); therefore, red muscles could contain less Ca2+. However, mitochondria, which are more abundant in red muscle, could be supplying free Ca2+ postmor- tem (Buege and Marsh, 1975; Mickelson, 1983). We determined water-soluble free Ca2+ at d 1 postmortem and found no differences (P > .05) among breed crosses (Table 4). There- fore, &+ content does not appear to be a limiting factor.

Guroff (1964) reported that Zn2+ inhibits CDP activity. Koohmaraie (199Ob) found that infusion of ovine carcasses with ZnCl2 in- hibited the aging response such that CDP-I activity did not decline by d 1 postmortem, as it normally does. We measured Zn2+ concen- trations; no differences (P > .05) were found (Table 4). However, because no si&icant breed moss differences were seen in fiber type

percentage, differences in overall mineral content would not be expected.

Another aspect of tenderization is the activity of the proteases involved in postmor- tem aging. One proteolytic system that may be involved is the lysosomal enzyme system. Cathepsins are located within the lysosomal membrane and may not be released during the normal storage period. LaCourt et al. (1986) found no indication of lysosomal rupture with > 3 wk of aging. When muscle samples were subjected to exogenous cathepsins B, D and L, degradation of myofibrillar proteins, including myosin and actin, occurred (Schwartz and Bird, 1977; Matsukura et al., 1981; Mikami et al., 1987; Ouali et al., 1988). However, Bandman and Zdanis (1988) failed to detect any myosin degradation from muscle aged for 21 d. It st i l l is controversial whether or not cathepsins are involved in postmortem tenderi- zation under normal conditions. Therefore, we determined whether cathepticenzyme activity differed between S A H and H x A. The lysosomes were isolated and then lysed to prevent their inhibition by hown cysteine protease inhibitors (Kirschke et al., 1983; Barrett, 1987). because as much as 70 to 75% of cathepticenzyme activity can be inhibited during extraction @therington et al., 1987). We found no significant breed-cross effects for cathepsin B or B + L specific activity (Table 4) at either d 1 or d 14. Therefore, it appears that cathepsins were not involved in our breed- cross differences in meat tenderization with 14 d of aging.

Another proteolytic system implicated in postmortem tenderization is the CDP system. The myofibrillar proteins hydrolyzed in vitro by CDP closely mimic changes observed under normal postmortem conditions (Dayton et al., 1976; Olson et al., 1977; Elgasim et al., 1985; Koohmaraie et al., 1988). Davey and Gilbert (1969) considered that meat aging was due to disruption and possible dissolution of Zdisk material, leading to a weakening of inter- myofibrillar linkages. With the addition of EDTA to chelate Ca2+, these changes did not occur. For activation, CDP-I requires 50 to 70

Ca2+ concentrations, but once autolyzed, it requires less Ca2+ for activity; CDP-JI requires 1 to 5 m~ Ca2+ for activity (MeUgren, 1980; Dayton et al., 1981). In living muscle, the very low level of free Ca2+ (<.01 mM) may regulate or limit indiscriminate destruction of the Z- disk by CDP in vivo. However, Nakamura

2724 WHIPpLE ET AL.

H x A

0 318 Sahiwal

518 Sahiwal

aJ 0

c 0 m 3004 0 0 . .= 200 > 0 150 m

.- C

aJ rn aJ v) 100

50

n

4-

2 n

T

" O h 24 h O h 24 h O h 24 h

CDP-I CDP-II inhibitor Figure 6. Longissimus calciumdependent protease-I, -II and CDP-inhibitor activities at < 1 and 24 h postmortem by

breed cross. Bars without a common superscript letter differ (P < .05).

(1973a.b) and Mickelson (1983) have shown that sarcoplasmic reticulum membranes lose their ability to sequester Ca2+ postmortem because of a drop in temperature and a depletion of ATP. Therefore, Ca2+ concentra- tions would increase postmortem. As indicated in Table 4, no breed-cross differences in Ca2+ content existed, and the range of 8.6 to 10.8 pg/g (.21 to .27 mM) was sufficient Ca2+ to activate CDP-I, but not CDP-II.

In addition to mimicking proteolysis seen in postmortem aging, CDP has been localized at the Z-disk and Sarcolemma and vaguely at the M-line in chicken skeletal muscle (Ishiura et al., 1980). In beef cardiac muscle, CDP was observed at the Z-disk and sarcolemma (Lane et al., 1985). Kleese et al. (1987) found CDP to be localized in crotaline (rattlesnake) stri- ated muscle at the I-band. Also, because of the cross-reaction between bovine and crotaline species, they concluded that both CDP and INH in remotely related organisms have very similar s m c m s .

We determined activities of CDP-I, -11 and INH within 1 h and at 24 h postmortem. No significant differences in activity/lOO g of muscle were seen for CDP-I and -II at < 1 and 24 h, and in INH at < 1 h (Figure 6). However, INH activity at 24 h was lower (P < .05) for H

x A (136.3 f 14.9) than for 3/8 SAH (195.6 f

maraie et al. (1987, 1989) observed decreases in CDP-I and INH activity with postmortem aging; decreased activity was associated with increased tenderness. Previous researchers have measured CDP and INH activities only on frozen tissue; however, because INH is susceptible to freezing (Otsuka and Goll, 1987; Koohmaraie, 199Oa), its true activity likely has been underestimated. In our study, CDP and INH activities were determined on fresh muscle, which provided new insight into the relationships among CDP, INH and tenderness. The initial CDP and INH activities were similar among breed crosses. Although breed crosses showed the same amount of CDP-I activity decline by 24 h, a significant differ- ence in tenderness was still observed Differ- ences in tenderness and INH activity at 24 h between SAH and H x A indicated that a relationship might exist between these two factors. Apparently, the CDP INH mechanism differs for SAH and H x A, resulting in a decreased ability of CDP-I to hydrolyze myofibrillar proteins in SAH.

For H x A, SDS-PAGE revealed that by d 1 desmin had been subjected to proteolysis, and by d 14 desmin could not be detected (Figure

19.3) and 5/8 SAH (208.8 f 14.8). Kwh-

BOS TAURUS AND BOS XNDICUS BEEP TENDERNESS 2725

H x A 3/8 SAH 5/8 SAH 14 0 1 14

Figure 7. SDS-PAGE of myofibrillar proteins isolated from longissimus muscle at < 1 4 1 and 14 d postmortem by breed cross.

7). instead, a 30,OOOdalton component was clearly evident. For 3/8 SAH, desmin was more intense at d 1 than for H x A, but by d 14, the 3/8 SAH gel resembled that of the H x A. However, in 5/8 S A H , desmin remained at d 14 and no 30,ooo-dalton component ap- peared. A 95,OOO-dalton component appeared by d 14 in all breed crosses, indicating that its appearance may not be associated with tender- ness. With beef stored at YC, the gradual disappearance of des& and the appearance of 110,000-, 95,000- and 30,ooOdalton polypep- tides has been reported (Koohmaraie et al., 1984; Xiong and Anglemier, 1989). MacBride and Parrish (1977) found that the major difference between tough and tender meat was associated with the presence of a 30,OOOdalton component. In a study compar- ing tenderness traits between Friesian and Friesian x Brahman bulls, the Z-disk region was more stable after aging in the Brahman crosses (F’urchas, 1972). These reports indicate that myofibrillar-protein hydrolysis is impor- tant in tenderization; this also is reflected in our MFI values (Figure 3). As the amount of protein hydrolysis increases, the myofibrils are more likely to fragment, thus increasing MFT The myofibrillar proteins that probably are responsible for the amount of fragmentation

are desmin and those associated with the Z - disk region.

Reduced protein hydrolysis appears to ac- count for less tender meat in S A H , the CDP INH mechanism may be influencing this process. We do not think that this is solely due to increased inhibitory action on CDP-I by INH in vivo. The binding of INH to CDP occurs in the presence of Ca2+, and it can be reversed by adding EDTA during extraction (DeMartino and Croall, 1985). We think that if there had been more inhibition CDP-I activity would have been greater at 24 h in SAH because less CDP-I would have been auto-

It is thought that INH is hydrolyzed by CDP (Imajoh and Suzuki, 1985; Mellgren et al., 1985,1986); however, if CDP and INH are not sequestered in close proximity to one another, hydrolization of INH likely could not occur. We found that INH activity in SAH did not decline as much by 24 h as it did in H x A. This may have been due to CDP and INH being in different locations. However, in studies involving skeletal and cardiac muscles of various species, no localization differences were found (Ishiura et al., 1980; Lane et al., 1985).

III the presence of sufficient ca2+, CDP may undergo autolysis (Hathaway et al., 1982;

lyzed.

2726 WHWPLE ET AL.

Coolican and Hathaway, 1984; Demartino et al., 1986; Crawford et al., 1987); this occurs more rapidly in the absence of a substrate or INH. "berefore, autolysis of CDP-I among breed crosses may have been different. Autoly- sis of CDP-I could have been greater in SAH, thus decreasing its ability to hydrolyze INH or myofibrillar proteins. However, because the rate of this process is dependent on the availability of a substrate or the presence of INH, a localization difference also is possible.

Another explanation could be that unknown protease(s) are involved. This has been pro- posed by other researchers to explain the loss in activity in CDP-I and INH (Vidalenc et al., 1983; Koohmaraie, 1990a). When CDP-I was inhibited by ZnCl2 infusion, thus preventing autolysis, Koohmaraie (199Ob) proposed that a protease possibly hydrolyzed CDP-I between d 1 and d 14 postmortem. Also, no change was detected in INH activity with ZnC12 infusion; this adds credence to the idea that CDP hydrolyzes INH. Therefore, unknown pro- tease(s) may be preventing CDP-I from hydrolyzing INH and myofibrillar proteins in SAH. Also, protease(s) could be present that hydrolyze INH in H x A but not in SAH. This would explain the decline in INH activity and, thus, lower the availability of INH to inhibit CDP-I protein hydrolysis in H x A but not in SAH.

From this discussion, it is apparent that much is unknown about the interaction be- tween CDP and INH. Our study suggests that there is a relationship between this mechanism and postmortem tenderization.

Implications

Less tender longissimus muscles of Bos indicus cattle compared with Bos raurus cattle apparently was due to reduced postmortem proteolysis of myofibrillar proteins in Bos indicus cattle. This reduced proteolysis was associated with higher activity of calcium- dependent protease inhibitor in Bos indicus cattle. Limited published research to date has examined the activity of calciumdependent protease or its inhibitor as a possible explana- tion for less tender meat in Bos indicus cattle than in Bos taurus cattle. Because aging Bus indicus longissimus steaks for 14 d did not result in acceptable tenderness, other tenderiza- tion techniques may be necessary. The reasons that Bos indicus cattle have higher inhibitor

activity and the mechanism(s) involved in its regulation are unknown. Therefore, more re- search is needed to understand and possibly control the mechanism(s).

Literature Cited

AOAC. 1975. Official Methods of Analysis (11th Ed.). Association of Officinl Analytical Chemists, Washing- ton, Dc.

AMSA. 1978. Guidelines for cookery and sensory evalua- tion of meat. American Meat Science Association, Chicago, IL.

Ashmore, C. R. and L. Doerr. 1971. Comparative aspects of muscle fiber types in different species. Exp. Neurol. 31:408.

Bandman, E. and D. Zdanis. 1988. An immunological method to assess protein degradation in post-mortem muscle. Meat Sci. 221.

Barrett, A. J. 1987. The cystatins: A new class of peptides inhibitors. TIBS 12:193.

Bechet, D., A. Obled and C. Deval. 1986. Species variations amongst proteinases in liver lysosomes. Biosci. Rep. 6: 991.

Bendall, J. R 1973. In: G. H. Bourne (Ed.) Structure and Function of Muscle. Vol. 2. p 243. Academic Press, New York.

Bergmas I. and R. Loxl9. 1963. Two improved and simplified methods for the spectrophotometric deter- mination of hydroxyproline. Anal. Chem. 35:1%1.

Bouton, P. E., F. D. Carrol, P. V. Harris and W. R Shorthose. 1973. Influence of pH and fiber contraction state upon factors affecting the tenderness of bovine muscle. J. Food Sci. 38:404.

Buege, D. R. and B. B. Marsh. 1975. Mitochondrial calcium and postmortem muscle shortening. Biochem. Bi- Ophys. Res. Commun. 65:478.

CalLins, C. R., T. R. Dutson, G. C. Smith, Z. L. Carpenter and G. W. Davis. 1981. Relationship of fiber type composition to marbling and tenderness of bovine muscle. J. Food Sci. 46:708.

Cole, J. W., C. B. Ramsey, C. S. Hobbs and R. S. Temple. 1963. Effects of type and breed of British, zebu, and dairy cattle on production carcass composition, and palatability. J. Dairy Sci. 2 1 1 3 8 .

Coolican, S. A. and D. R. Hathaway. 1984. Effect of L- alpha-phosphatidylinositol on a vascular smooth muscle calcium-dependent protease. J. Biol. Chem. 259:11627.

Crawford, C., A. C. Willis and J. Gagnon. 1987. The effects of autolysis on the strocture of chicken calpain II. Biochem. J. 248579.

Cmckctt, J. R., R. S. Baker, Jr., J. W. Carpenter and M.

teristics of calves sired by Continental, Brahman and Brahman-derivative sires in subtropical Florida. J. Anim. Sci. 49900.

Cross. H. R., Z. L. Carpenter and G. C. Smith. 1973. Effects of intramuscular collagen and elastin onbovme muscle tenderness. J. Pood Sci. 38998.

Cross, H. R., R. L. West and T. R. Dutson. 1981. Comparison of methods for meaSuring sarcomere 1cngthinbttfscmitendinososmuscle.Mcat Sci. 5261.

Crouse, J. D., L. V. Cundif€, R M. Koch, M. Koohmaraie and S. C. Seideman. 1989. Comparisons of Bos indicus andBos r a m s inheritance on carcass beef characteris-

KOga. 1979. Reweaniog, feedlot and ~ ~ K W S S charac-

BOS TAURUS AND BOS INDICUS BEEF TENDERNJSS 2727

tics and meat palatability. J. Anim. Sci. 672661. Crouse, J. D.. S. C. Seideman and L. V. Cundiff. 1987. The

effect of carcass electrical stimulation on meat obtained from Bos indim and Bos t a m s cattle. J. Food Qual. 10:407.

Culler, R D., F. C. Parrish, Jr., G. C. Smith and H. R Cross. 1978. Relationship of myofibril fragmentation index to certain chemical, physical and sensory characteristics of bovine. longissimus muscle. J. Food Sci. 431177.

Davey, C. L. and K. V. Gilbert. 1969. Studies in meat tenderness. 7. changes in the fme structure of meat during aging. J. Food Sci. 3469.

Davis, G. W., G. C. Smith, 2. L. Carpenter, T. R. Dutson and H. R. Cross. 1979. Tenderness variations among beef steaks from carcasses of the same USDA quality grade. J. Anim. Sci. 49103.

Dayton, W. R., W. J. Reville, D. E. G o U and M. H. Stroma. 1976. A calcium-activated protease possibly involved in myofibrillar protein turnover. Partial characterizn- tion of the purified enzyme. Biochemistry 15:2159.

Dayton, W. R., J. V. Schollmeyer, R. A. Lepley and L. R. Cortes. 1981. A calcium-activated protease involved in myofibrillar protein turnover. Isolation of a low- calcium raqUiring form of the protease. Biochim. Biophys. Acta 65948.

DeMartino, G. N. and D. E. Croall. 1985. Calcium- dependent proteases from liver and heart. In: E. A. Khairallah (Ed.) Intracellular Protein Catabolism. pp 117-126. Alan R Lis, New York.

DeMartino, G. N., C. A. Huff and D. E. Croall. 1986. Autoproteolysis of the small subunit of calcium dependent protease-II activates and regulates protease activity. J. Biol. Chem. 261:12047.

Dikeman, M. E., K. N. Nagele, S. M. Myers, R. R. Schalles, D. H. Kropf, C. L. Kastner and F. A. Russo. 1985. Accelerated versus conventional beef production and processing. J. Anim. Sci. 61:137.

Dransfield, E. 1977. Intramuscular composition and texture of beef muscles. J. Sci. Food Agric. 28:833.

M e l d , E., R.C.D. Jones and H.J.H. MacFie. 1981. Quantifying changes in tenderness during storage of beef. Meat Sci. 5:131.

Elgasim, E. A,, M. Koohmaraie, A. F. Anglemier, W. W. Kennick and E. A. Elkhalifa. 1985. The combined effects of the calcium-activated factor and cathepsinD on skeletal muscle. Food Microstruct. 455.

Etherington, D. J., MAJ. Taylor and E. W i e l d . 1987. Conditioning of meat from different species. Relation- ships between tenderizing and the levels of Cathepsin B. Cathepsin L, Calpain I, Calpab II and glucoronidase. Meat Sci. 201.

Gauthier, G. F. 1970. The ultrasound of three fiber types in mammalian skeletal muscle. In: E. J. Bnskey, R. G. Cassens and B. B. Marsh (Ed.) Physiology and Biochemishy of Muscle as a Food. Univ. of Wisconsin Press, Madison.

Guroff, G. 1964. A neutml, calcium-activated proteinase from the soluble fractions of rat brain. J. Biol. Chem. 239: 149.

Hall, J. B. and M. C. Hunt. 1982. Collagen solubility of A- mahuity bovine longissimus muscle as affected by nutritional regimen. J. Anim. Sci. 55:321.

Harris, P. V. 1976. Slructural and other aspects of meat tenderness. J. Texture Stud. 7:49.

Hathaway, D. R., D. K. Werth and J. R. Haeberle. 1982. Limited autolysis reduces the calcium requirement of a smooth muscle calcium-activated protease. J. Biol.

Chem. 257972. Hill, F. 1%6. The solubility of intrammcular collagen in

meat animals of various ages. J. Food Sci. 31:161. Hunt, M. C. and H. B. Hedrick. 1977. Proffie of fiber types

and related properties of five bovine muscles. J. Food Sci. 42:513.

Imajoh, S. and K. Suzuki. 1985. Reversible interaction between calcium-activated neutral protease (CANP) and its endogenous inhibitor. FBBS Lett. 187:47.

Ishiura, S., H. Sugita, I. Nonaka and K. Imahori. 1980. Calcium-activated neutral protease, its localization in the myofibril, especially at the Zband. J. Biochem. (Tokyo) 87:343.

Kirschke, H., L. Wood, P. J. Roisen and J.W.C. Bird. 1983. Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle. Biochem. J. 214 871.

Kleese. W. C., D. E. Goll, T. Edmunds and J. D. Shannon. 1987. Immunofluorescent localization of the calcium- dependent proteinase and its inhibitor in tissues of Crotalus atrox. J. Exp. Zool. 241:277.

Koch, R. M., J. D. Crouse, M. E. Dikeman. L. V. Cundiff and K. E. Gregory. 1988. Effects of marbling on sensory panel tenderness inBos rawus and Bos indicus crosses. J. Anim. Sci. 66(Suppl. 1):305(Abstr.).

Koch, R M., M. E. Dikeman and J. D. Crouse. 1982. Characterization of biological types of cattle (Cycle III). III. Carcass composition, quality and palatability. J. Anim. Sci. 5435.

Koohmaraie, M. 199Oa. Quantification of Caz+-dependent protease activities by hydrophobic and ion-exchange chromatography. J. Anim. Sci. 68:659.

Koohmaraie, M. 199Ob. Inhibition of postmortem tenderiza- tion in ovine carcasses through infusion of zinc. J. Anim. Sci. 68:1476.

Koohmaraie, M., A. S. Babiker, R. A. Merkel and T. R. Dutson. 1988. Role of calcium-dependent proteases and lysosomal enzymes in postmortem changes in bovine skeletal muscle. J. Food Sci. 53:1253.

Koohmaraie, M., J. D. Crouse and H. J. Mersmann. 1989. Acceleration of postmortem tenderization in ovine carcasses through infusion of calcium chloride: Effect of concentration and ionic strength. I. Anim. Sci. 67: 934.

Koohmaraie, M., W. H. Kennick, E. A. Elgasim and A. F. Anglemier. 1984. Effects of postmortem storage on muscle protein degradation: Analysis by SDS-poly- acrylamide gel electrophoresis. J. Food Sci. 49292.

Koohmaraie, M., S. C. Seidemau, J. E. Schollmeyer, T. R. Dutson and J. D. Crouse. 1987. Effect of postmortem storage on calcium-dependent proteases, their inhibitor and myofibril fragmentation. Meat Sci. 19187.

Lacourt, A., A. Obled, C. DeVal, A. Ouali and C. Valin. 1986. Postmortem localization of lysosomal peptide hydrolase, cathepsin B. In: V. Turk (Ed.) Cysteine Proteinases and their Inhibitors. pp 239-248. Walter de Gruyter and Co., Berlin, NY.

Laemmli, U. K. 1970. Cleavage of storage proteins during the assembly of the head bacteriophage T4. Nature 227:680.

Lane, R. D., R. L. Mellgren and M. T. Mericle. 1985. Subcellular localization of bovine hcart calcium- dependent protease inhibitor. J. Mol. Cell. Cardiol. 17 863.

MacBride, M. A. and F. C. Parrish, Jr. 1977. The 30,000-dalton component of tender bovine longis- simus muscle. J. Food Sci. 46:1627.

Markwell, M. K., S. M. Haas, L. L. Bieber and N. E. Tolbert.

2728 WHIPPLE ET AL.

1978. A modification of the Lowry procedure to simpllfy protein determination in membrane and lipoprotein samples. Anal. Biochem. 87206.

Marsh, B. B. and N. G. Leet. 1966. Studies in meat tenderness. III. The effects of cold shortening on tenderness. J. Food Sci. 31:450.

Marsh, B. B., J. V. Lochner, G. Takahashi and D. D. Kragness. 1981. Effects of early postmortem pH and temperature on beef tenderness. Meat Sci. 5:479.

Matsukura, U., A. Okitaoi, T. Nishimura and H. Kato. 1981. Mode of degradation of myofibrillar proteins by an endogenous protease. cathepsin L. Biochim. Biophys. Acta 66241.

McKeith, F. K., D. L. DeVol, R. S. Miles, P. J. Bechtel and T. R. Carr. 1985a. Chemical and sensory properties of thirteen major beef muscles. J. Food Sci. 50:869.

McKeith, F. K.. J. W. Smith, G. C. Smith, T. R. Dutson and Z. L. Carpenter. 1985b. Tenderness of major muscles from three breed-types of cattle at different times-on- feed. Meat Sci. 13151.

Mellgren, R. L. 1980. Canine cardiac calcium-dependent proteases: Resolution of two forms with different requirements for calcium. FEBS Lett. 109:129.

Mellgren, R L., R. D. Lane, M. G. Hegazy and E. M. Reimann. 1985. The specifc inhibitor of calcium- dependent protease. A heat-stable phosphoprotein with properties similar to the inhibitor of phosphoprotein phosphates. Adv. Protein Phosphatases 1259.

Mellgren, R. L., M. T. Mericle and R D. Lane. 1986. Proteolysis of the calcium-dependent protease iuhib- itor by myocardial calciumdependent protease. Arch. Biochim. Biophys. 246:233.

Melton, C. C., M. E. Dikeman, H. I. Tmna and D. H. Kropf. 1975. Histochemical relationships of muscle biopsies with bovine muscle quality and oomposition. I. Anim. Sci. m451.

Mickelson, J. R. 1983. Calcium transport by bovine skeletal muscle mitochondria and its relationship to postmor- tem muscle. Meat Sci. 9:205.

Mikami, M., A.H. Whitiug.M.A.J. Taylor, R.A. Maciewicz and D. J. Etherington. 1987. Degradation of myofibrils from rabbit, chicken and beef by cathepsin L and lysosomal lysates. Meat Sci. 21:81.

Moody, W. G.. D. A. Tichenor. J. D. Kemp and J. D. Fox. 1970. Effects of weight, castration and rate of gain on muscle fiber and fat cell diameter in two ovine muscles. J. Anim. Sci. 31:676.

Naknmura, R 1973a. Rstimation of water-extractable calcium in chicken breast muscle by atomic absorp- tion. Anal. Biochem. 53:531.

Nakamura, R. 1973b. Factors associated with postmortem increase in extractable calcium m chicken breast muscle. J. Food Sci. 38:1113.

and C. J. Pierson. 1984. Castration and sire effects on carcass traits, meat palatability and muscle fiber characteristics in Angus cattle. J. Anim. Sci. 59:981.

Olson, D. G., P. C. Panish, Jr., W. R Dayton and D. E. Goll. 1977. Effect of postmortem storage and calcium activated factor on the myofibrillar proteins of bovine skeletal muscle. J. Food Sci. 42117.

Olson, D. G., F. C. Panish, Jr. and M. H. Stromer. 1976. Myofibril fragmentation and shear resistance of three bovine muscles during postmortem storage. J. Food Sci. 41:1036.

Otsuka, Y. and D. E. Goll. 1987. purification of the calcium- dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar calcium- dependent proteinase. I. Biol. Chem. 2625839.

Ouali, A., E. Dufour, A. Obled, C. Deval and C. Valin. 1988. Effect of muscle proteinases on fast and slow myosins. Relationship with postmortem proteolysis in muscles of variable contractility. Reprod. Nutr. Dev. 289339.

pluchas, R. W. 1972. The relative importance of some determinants of beef tenderness. J. Food Sci. 37:341.

SAS. 1985. SAS User’s Guide: Statistics. SAS Inst., Inc., W, NC.

Schw&, W. N. and J.W.C. Bird. 1977. Degradation of myofibrillar proteins by cathepsins B and D. Biochem. J. 167:811.

Seideman, S. C. and J. D. Crouse. 1986. The effects of sex condition, genotype and diet on bovine muscle fiber characteristics. Meat Sci. 1755.

Seideman, S. C., M. Koohmaraie and J. D. Crouse. 1987. Factors associated with tenderness in young beef. Meat Sci. 20281.

Solomon, M. B. and M. C. Dunn. 1988. Simultaneous histochemical determination of three fiber types in single sections of ovine, bovine and porcine skeletal muscle. J. Anim. Sci. 66255 .

Tatum, I. D., G. C. Smith, B. W. Berry, C. E. Murphey, F. L. Williams and Z. L. Carpenter. 1980. Carcass character- istics, time on feed and cooked beef palatab~ty attributes. J. Anim. Sci. 50833.

Umuh, I. A, D. G. Gray and M. E. Dikeman. 1986. Implanting young bulls with Zeranol from birth to four slaughter ages: E. Carcass quality, palatability and muscle-collagen characteristics. J. Anim. Sci. 62388.

USDA. 1976. official United States Standards for Grades of Carcass Beef. USDA A@. Marketing Sew.. Wash-

Vidalenc, P., P. Cottin, N. Merdaci and A. Ducastaing. 1983. Stability of two calcium-dependent neutral proteinases and lhei specific inhibitor during postmortem storage ofrabbit skeletalmuscle. J. Sci. Food Agric. 34:lMl.

Xionn. X. L. and A. P. Annlemier. 1989. Gel electrophoresis

ington, Dc.

-. analysis of the protei changes in ground beef stored at

Ockerman, H. W., D. Jaworek, B. VanStavem, N. Parrett 2 C. J. Food Sci. 54:287.